Compositions comprising sulforaphane or a sulforaphane precursor and a mushroom extract or powder

ABSTRACT

The present disclosure relates to the synergistic combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, a cofactor of the enzyme, and a glucan. The present disclosure also relates to the synergistic combination of sulforaphane or a derivative thereof and a glucan. The present disclosure also relates to the synergistic combination of a broccoli extract or powder and a glucan. The glucan may be a β-glucan. The glucan may be provided in a mushroom extract or powder selected from one or more of a maitake, a shiitake, or a reishi mushroom. Compositions and methods relating to these combinations are described.

This application is a continuation patent application of U.S. patentapplication Ser. No. 15/244,374 filed on Aug. 23, 2016, which is acontinuation patent application of U.S. patent application Ser. No.14/586,765 filed on Dec. 30, 2014 (now U.S. Pat. No. 9,421,183) which isa continuation of International Application No. PCT/US2013/049248 filedon Jul. 3, 2013, the contents of each of which are incorporated hereinby reference in their entirety, and also claims priority to thefollowing applications, each of which is incorporated by reference inits entirety: U.S. Provisional Patent Application No. 61/668,328, filedon Jul. 5, 2012; U.S. Provisional Patent Application No. 61/668,342,filed on Jul. 5, 2012; U.S. Provisional Patent Application No.61/668,386, filed on Jul. 5, 2012; U.S. Provisional Patent ApplicationNo. 61/668,396, filed on Jul. 5, 2012; U.S. Provisional PatentApplication No. 61/668,364, filed on Jul. 5, 2012; U.S. ProvisionalPatent Application No. 61/668,374, filed on Jul. 5, 2012; and U.S.Provisional Patent Application No. 61/794,417, filed on Mar. 15, 2013.

FIELD OF THE INVENTION

The present invention relates to the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and a mushroom (preferably maitake,shiitake, or reishi mushroom) extract or powder. The present inventionalso relates to the combination of sulforaphane or a derivative thereofand a mushroom (preferably maitake, shiitake, or reishi mushroom)extract or powder. The present invention also relates to the combinationof a broccoli extract or powder and a mushroom (preferably maitake,shiitake, or reishi mushroom) extract or powder. The present inventionprovides compositions and methods relating to these combinations.

BACKGROUND OF THE INVENTION

The use of natural products is becoming increasingly popular with humansand companion animals. Some of these natural products are beingincorporated into dietary supplements and medical foods. There is a needin the art for supplements which are useful as chemoprotective and/orantioxidant agents. In addition, there is a need in the art forpharmaceutical compositions and dietary supplements which are useful forconditions and disorders associated with the breast.

Chemoprotection through the use of natural products is evolving as asafe, effective, inexpensive, easily accessible, and practical means toprevent or reduce the occurrence of many conditions affecting humans anddomesticated animals. It is known that carcinogens which can damagecells at the molecular level are often ingested and inhaled as non-toxicprecursors. These non-toxic precursors may then convert intocarcinogenic substances in the body. Chemoprotective agents, such asnatural substances which can activate detoxifying enzymes or theirco-factors, can counteract and allow for the elimination or potentiatethe other naturally existing defenses such as the immune system.

Some natural products have antioxidant activity. Oxidative stress playsa major role in aging, the progression of neurodegenerative diseases aswell as physiological trauma, such as ischemia. Antioxidant agents canreduce or inhibit the oxidation of vital biomolecules and may play arole in treating, preventing, or reducing the occurrence of cancer,coronary heart disease, stroke, and neurodegenerative diseases,Alzheimer's Disease, dementia, and stroke are examples of conditionsaffected by oxidative stress.

Cancers are largely thought to be a consequence of exposure toenvironmental challenges—whether from within (i.e.—estrogen,progesterone hormones) or externally (i.e.—bisphenol A (BPA) fromplastic)—and chronic inflammation. Fortunately, the damage fromenvironmental challenges can be negated via a complex network of PhaseII chemoprotective enzymes found in many cell types of our body. It iswell known that estrogens and their metabolites can lead to theproliferation of breast tissue and tumors. Worse, the quinone estrogenmetabolites have the capacity to enter the breast tissue and migrateinto the nucleus of ductal and glandular epithelial cells. There, theybind to DNA forming quinone estrogen DNA adducts which lead todownstream mutations. These mutations are thought to be responsible forthe very foundation of a tumor: cancer initiation. Fortunately, aparticular phase II enzyme, NAD(P)H:quinone oxidoreductase (NQO1) cantake dangerous and highly reactive quinone estrogens and metabolize themto inert chemicals that can readily be removed from the body. Thus, amajor mechanism to decrease cancer incidence is to induce protectivePhase II enzymes including NQO1. Increased levels of NQO1 can beeffective at treating, preventing, repairing, reducing the occurrenceof, decreasing the symptoms associated with any conditions which areresulting from high levels of quinone estrogens. Examples of quinoneestrogens include but are not limited to catechol quinones of estrogen.Quinone estrogens are described in the following references, each ofwhich is incorporated by reference in its entirety: Nutter et al. ChemRes Toxicol, 1994, 7:23-28; Cavalieri et al. Ann NY Acad Sci, 2006;1089:286-301; Bolton et al. Chem Res Toxicol, 2008, 21(1):93-101; andCavalieri et al., Biochimica et Biophysica Acta, 2006, 1766:63-78.

An example of a natural product thought to have chemoprotective andantioxidant properties is sulforaphane. Sulforaphane is an organosulfurcompound which is also known as 1-isothiocyanato-4-methylsulfinylbutane.The sulforaphane precursor, glucoraphanin, can be obtained fromvegetables of the Brassicaceae family, such as broccoli, brusselsprouts, and cabbage. However, copious amounts of vegetables must beconsumed in order to obtain levels adequate for chemoprevention.Glucoraphanin is converted into sulforaphane by a thioglucosidase enzymecalled myrosinase, which occurs in a variety of exogenous sources suchas Brassicaceae vegetables and endogenously in the gut microflora.However, upon ingestion of glucoraphanin, not all animals are capable ofachieving its conversion to sulforaphane, most likely due to variationsin microflora populations and overall health. In addition, in acidicenvironments such as the stomach, glucoraphanin can be converted toinert metabolites. The active metabolite, sulforaphane induces nuclearerythroid-2-related factor (Nrf2) which, in turn, upregulates theproduction of Phase II detoxification enzymes and cytoprotective enzymessuch as glutathione S-transferases, NAD(P)H:quinone oxidoreductase(NQO1), and heme-oxygenase-1 (HO-1). Sulforaphane has been thought toinduce the production of these enzymes without significantly changingthe synthesis of P-450 cytochrome enzymes. The upregulation of Phase IIenzymes is thought to play a role in a variety of biological activities,including the protection of the brain from cytotoxicity, the protectionof the liver from the toxic effects of fat accumulation, and thedetoxification of a variety of other tissues.

Sulforaphane and its precursor glucoraphanin have been studiedextensively. Shapiro et al. (Nutrition and Cancer, (2006), Vol. 55(1),pp. 53-62) discuss a clinical Phase I study determining the safety,tolerability, and metabolism of broccoli sprout glucosinolates andisothiocyanates. Shapiro et al. discuss a placebo-controlled,double-blind, randomized clinical study of sprout extracts containingeither glucosinolates such as glucoraphanin or isothiocyanates such assulforaphane in healthy human subjects. The study found thatadministration of these substances did not result in systematic,clinically significant, adverse effects. Ye et al., (Clinica ChimicaActa, 200, 316:43-53) discuss the pharmacokinetics of broccoli sproutisothiocyanates in humans.

A number of mushrooms have been used or studied for their medicinaleffects. These “medicinal mushrooms” are thought to have beneficialproperties, such as antiviral, antimicrobial, anticancer,antihyperglycemic, and/or anti-inflammatory activity. Examples ofmedicinal mushrooms include maitake, shiitake, reishi, cremini, almond,chestnut, wood ear, cloud ear, porcini, ink cap, yarta gunbu, enokitake,shemeji, tiger milk, morel, bamboo, golden oyster, pink oyster, kingoyster, hiratake, cauliflower, white jelly, golden jelly, matsutake,Mexican truffle, and straw mushrooms.

Maitake mushrooms (Grifola frondosa) are edible mushroom consumed widelyas food and used in traditional medicine to enhance immune function andto treat cancer. Maitake mushrooms, which contain glucans, are thoughtto have beneficial properties, such as antitumor and immunomodulatoryeffects. There exist standardized extracts from maitake mushroom thatcontain as active ingredients glucans such as protein-boundbeta-glucans. Beta 1,6-glucan, a protein bound polysaccharide, has beenidentified as an active constituent in maitake mushrooms. Maitakemushrooms have been demonstrated to have antitumor effects, inhibitingtumor metastasis in vitro. In one study, tumor regression or significantimprovements in symptoms were observed in half of the subjects usingmaitake extract. In a study of postmenopausal breast cancer patients,oral administration of maitake extract was shown to haveimmunomodulatory effects.

Shiitake mushrooms (Lentinula edodes) are edible mushrooms native toEast Asia. Shiitake mushrooms contain mycochemicals, which arepostulated to have antiviral, antibiotic, anti-inflammatory,antihypertensive and anticarcinogenic effects. This is thought to belargely a result of glucans, both alpha and beta glucans. Some shiitakemushroom extracts have alpha glucan content greater than 40%.Additionally, lentinan (1,3 beta-D-glucan), a polysaccharide isolatedfrom shiitake, has been well studied and is thought to play a role inshiitake's beneficial effects. It has been shown to have anticancereffects in colon cancer cells, which may be due to its ability tosuppress cytochrome P450 1A enzymes that are known to metabolizepro-carcinogens to active forms. Lentin, the protein component, hasstrong antifungal properties and has been found to inhibit proliferationof leukemic cells and suppress the activity of human immunodeficiencyvirus-1 reverse transcriptase.

Reishi mushrooms (Ganoderma lucidum), also known as lingzki mushrooms,are edible mushrooms found in East Asia. Reishi mushrooms are thought tohave anti-tumor, anti-cancer, immunomodulatory, and immunotherapeuticeffects. Reishi mushrooms have a number of components which are thoughtto contribute to its activity, including glucan, such as beta-glucan,canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

Baker's yeast (Saccaromyces cerevisiae) can be a source of glucans, inparticular, beta-glucans. The active components of Baker's yeast can beextracted in a number of ways, such as the methods described in Bacon etal. Biochem J, 1969, 114(3): 557-567, U.S. Pat. Nos. 7,803,605;5,702,719; and 8,323,644, each of which is incorporated by reference inits entirety.

Glucans are described in the following references, which are eachincorporated by reference in its entirety: Vetvicka et al. Endocr MetabImmun Disord Drug Targets, 2009, 9(1):67-75, and Vetvicka et al. J MedFood, 2008: 11(4): 615-622.

Zhang et al. (Proc. Natl. Acad. Sci., (1994), Vol. 91, pp. 3147-3150)discusses a study in Sprague-Dawley rats to determine theanticarcinogenic activities of sulforaphane and structurally relatedsynthetic norbornyl isiothiocyanates. The study determined thatadministration of sulforaphane was effective in blocking the formationof mammary tumors.

Cornblatt et al. (Carcinogenesis, (2007), Vol. 38(7): pp. 1485-1490)discusses a study in Sprague-Dawley rats to determine the effect ofsulforaphane in chemoprevention in the breast. The study determined thatoral administration of either sulforaphane resulted in a 3-fold increasein NAD(P)H:quinone oxidoreductase (NQO1) enzymatic activity and a 4-foldelevated immunostaining of the heme oxygenase-1 (HO-1) enzyme in themammary epithelium.

Munday et al. (Cancer Res, (2008), Vol. 68(5): pp. 1593-1600) discussesa study regarding the effects of a freeze-dried aqueous extract ofbroccoli sprouts on bladder cancer development in rats. The study foundthat administration of the broccoli sprout extract resulted in asignificant induction of glutathione S-transferase and NAD(P)H:quinoneoxidoreductase 1 in the bladder, which are enzymes having protectiveactivity against oxidants and carcinogens.

Fang et al. (J Altern Complem Med, (2006), Vol. 12(2): pp. 125-132)discloses a study determining the antiproliferative effect of an ethylacetate fraction of shiitake mushrooms on human breast carcinoma celllines (MDA-MB-453 and MCF-7), a human nonmalignant breast epithelialcell line (MCF-10F), and two myeloma cell lines (RPMI08226 and IM-9).The study found that the inhibition of growth of tumor cells by thecomponents in shiitake mushrooms may result from the induction ofapoptosis.

Kim et al. (J Med Food, (2007), Vol. 10(1): pp. 25-31) discloses a studyinvestigating the activation of natural killer (NK) cells and anticancereffects of an exo-biopolymer from rice bran cultured from Lentinusedodes. The study found that the exo-biopolymer may be effective forpreventing and/or treating cancer through natural killer cellactivation.

Louie et al. (BJUI, (2009), Vol. 153(9): pp. 1215-1221) discusses thesynergistic effect of the combination of interferon-α and maitakemushroom D-fraction (PDF), a bioactive mushroom extract on anticanceractivity of interferon-α in bladder cancer T24 cells in vitro.

Masuda et al. (Biol. Pharm. Bull. (2008), Vol. 31(6): pp. 1104-1108)discusses a study assessing the anti-metastatic activity of a fractionof maitake mushrooms in a murine model of lung metastasis. The studyfound that the fraction inhibited tumor metastasis by activation ofnatural killer cells and antigen-presenting cells (APCs) and suppressingadhesion molecules such as ICAM-1, leading to the inhibition of tumorcell adhesion to vascular endothelial cells.

European Patent Application No. 2 213 280 discloses formulationscomprising glucosinolates such as glucoraphanin, and myrosinase, whereinthe formulation is encapsulated or coated.

All references cited herein are incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising: (i) asulforaphane precursor, preferably glucoraphanin; (ii) an enzyme capableof converting the sulforaphane precursor to sulforaphane, preferably aglucosidase enzyme, more preferably a thioglucosidase enzyme, and mostpreferably myrosinase; (iii) an enzyme potentiator or cofactor,preferably ascorbic acid; and (iv) a mushroom (preferably maitake,shiitake, or reishi mushroom) extract or powder. The present inventionalso provides a method of treating, preventing, reducing the occurrenceof, decreasing the symptoms associated with, and/or reducing secondaryrecurrences of, cancer, in particular breast cancer, prostate cancer,colon cancer, lung cancer, and bladder cancer in a subject, comprisingadministering to the subject: (i) a sulforaphane precursor, (ii) anenzyme capable of converting the sulforaphane precursor to sulforaphane,(iii) an enzyme potentiator, and (iv) a mushroom (preferably maitake,shiitake, or reishi mushroom) extract or powder. The present inventionalso provides a method of increasing levels or increasing geneexpression of NAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject,comprising administering to the subject: (i) a sulforaphane precursor,(ii) an enzyme capable of converting the sulforaphane precursor tosulforaphane, (iii) an enzyme potentiator, and (iv) a mushroom(preferably maitake, shiitake, or reishi mushroom) extract or powder.The present invention also provides a method of treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of a disease or conditionassociated with elevated levels of quinone estrogen, comprisingadministering to the subject: (i) a sulforaphane precursor, (ii) anenzyme capable of converting the sulforaphane precursor to sulforaphane,(iii) an enzyme potentiator, and (iv) a mushroom (preferably maitake,shiitake, or reishi mushroom) extract or powder.

The present invention provides a composition comprising: (i)sulforaphane or a derivative thereof, and (ii) a mushroom (preferablymaitake, shiitake, or reishi mushroom) extract or powder. The presentinvention also provides a method of treating, preventing, reducing theoccurrence of, decreasing the symptoms associated with, and/or reducingsecondary recurrences of, cancer, in particular breast cancer, prostatecancer, colon cancer, lung cancer, and bladder cancer in a subject,comprising administering to the subject: (i) sulforaphane or aderivative thereof, and (ii) a mushroom (preferably maitake, shiitake,or reishi mushroom) extract or powder. The present invention alsoprovides a method of increasing levels or increasing gene expression ofNAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject, comprisingadministering to the subject, comprising administering to the subject:(i) sulforaphane or a derivative thereof, and (ii) a mushroom(preferably maitake, shiitake, or reishi mushroom extract) or powder.The present invention also provides a method of treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of a disease or conditionassociated with elevated levels of quinone estrogen, comprisingadministering to the subject: (i) sulforaphane or a derivative thereof,and (ii) a mushroom (preferably maitake, shiitake, or reishi mushroom)extract or powder.

The present invention provides a composition comprising: (i) a broccoliextract or powder, and (ii) a mushroom (preferably maitake, shiitake, orreishi mushroom) extract or powder. The present invention also providesa method of treating, preventing, reducing the occurrence of, decreasingthe symptoms associated with, and/or reducing secondary recurrences of,cancer, in particular breast cancer, prostate cancer, colon cancer, lungcancer, and bladder cancer in a subject, comprising administering to thesubject: (i) a broccoli extract or powder, and (ii) a mushroom(preferably maitake, shiitake, or reishi mushroom) extract or powder.The present invention also provides a method of increasing levels orincreasing gene expression of NAD(P)H:quinone oxidoreductase 1 (NQO-1)in a subject, comprising administering to the subject, comprisingadministering to the subject: (i) a broccoli extract or powder, and (ii)a mushroom (preferably maitake, shiitake, or reishi mushroom) extract orpowder. The present invention also provides a method of treating,preventing, reducing the occurrence of, decreasing the symptomsassociated with, and/or reducing secondary recurrences of a disease orcondition associated with elevated levels of quinone estrogen,comprising administering to the subject: (i) a broccoli extract orpowder, and (ii) a mushroom (preferably maitake, shiitake, or reishimushroom) extract or powder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the conversion of glucoraphanin at 38° C.without ascorbic acid, as described in Example 4.

FIG. 2 is a graph showing the conversion within about 10 minutes at 38°C. as a function of ascorbic acid concentration, as described in Example4.

FIG. 3 is a graph showing the conversion to sulforaphane within 30minutes at 38° C. and 1 mM ascorbic acid, as described in Example 4.

FIG. 4 is a graph showing the conversion of glucoraphanin tosulforaphane in simulated intestinal fluid, as described in Example 5.

FIG. 5 is a graph showing the results of the experiment described inExample 6.

FIG. 6 is a graph showing the results of the experiment described inExample 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and a mushroom (such as maitake,shiitake, or reishi mushroom) extract or powder. The present inventionalso relates to the combination of sulforaphane or a derivative thereofand a mushroom (such as maitake, shiitake or reishi mushroom extract orpowder. The present invention also relates to the combination of abroccoli extract or powder and a maitake, shiitake, or reishi mushroomextract or powder. The present invention also relates to the use of amushroom extract or powder, with a mixture of one or more of thefollowing: sulforaphane precursor, sulforaphane or a derivative thereof,and broccoli extract. The present invention provides compositionsrelating to these combinations.

The present invention also provides methods comprising administeringthese combinations. In some embodiments, the combination may beadministered for treating, preventing, reducing the occurrence of,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of, cancer, in particular breast cancer, prostate cancer,colon cancer, lung cancer, and bladder cancer in a subject, comprisingadministering to the subject. In some embodiments, the combination maybe administered for increasing levels or increasing gene expression ofNAD(P)H:quinone oxidoreductase 1 (NQO-1) in a subject. In someembodiments, the combination may be administered for treating,preventing, reducing the occurrence of, decreasing the symptomsassociated with, and/or reducing secondary recurrences of a disease orcondition associated with elevated levels of quinone estrogen.

Sulforaphane is also known as 1-isothiocyanato-4-methylsulfinylbutane.Derivatives of sulforaphane include, but are not limited tosulfoxythiocarbamate analogues of sulforaphane, 6-methylsulfinylhexylisothiocyanate (6-HITC), and compounds which comprise the structure ofsulforaphane with different side chains and/or various lengths ofspacers between the isothiocyanato and sulfoxide groups. Examples ofderivatives of sulforaphane include those described in the followingreferences, each of which is incorporated herein by reference: Hu etal., Eur J Med Chem, 2013, 64:529-539; Ahn et al., Proc Nall Acad SciUSA, 2010, 107(21):9590-9595; and Morimistu et al., J. Biol. Chem. 2002,277:3456-3463, and Baird et al., Arch Toxicol, 2011, 85(4):241-272.

In some embodiments, the composition comprises sulforaphane or aderivative thereof, preferably sulforaphane, in an amount of about 1 μgto about 10 g, preferably about 3 μg to about 5 g, preferably about 5 μgto about 1000 mg, preferably about 7 μg to about 750 mg, more preferablyabout 10 μg to about 500 mg, and most preferably about 100 μg to about100 mg. In some embodiments, compositions suitable for human usecomprise about 1 mg to about 20 mg.

In some embodiments, the methods of the present invention compriseadministration of sulforaphane or a derivative thereof to a subject,preferably sulforaphane, in an amount of about 1 μg to about 10 g,preferably about 3 μg to about 5 g, preferably about 5 μg to about 1000mg, preferably about 7 μg to about 750 mg, more preferably about 10 μgto about 500 mg, and most preferably about 100 μg to about 100 mg. Insome embodiments wherein the subject is human, the method comprisesadministration of about 1 mg to about 20 mg. In some embodiments, themethods of the present invention comprise administration of sulforaphaneor a derivative thereof to a subject, preferably sulforaphane, in anamount of about 0.01 μg/kg to about 0.2 g/kg, preferably about 0.05μg/kg to about 0.07 g/kg, more preferably about 0.07 μg/kg to about 15mg/kg, more preferably about 0.1 μg/kg to about 11 mg/kg, and mostpreferably about 0.2 μg/kg to about 7 mg/kg. In some embodiments whereinthe subject is human, the method comprises administration of about 2μg/kg to about 2 mg/kg, and more preferably about 0.01 mg/kg to about0.3 mg/kg. The above amounts may refer to each dosage administration ora total daily dosage. The total daily dosage refers to the total amountof a compound or ingredient which is administered to a subject in atwenty-four hour period.

In some embodiments, the method comprises administration of more thanone of a sulforaphane or a derivative thereof. In some embodiments, thecompositions comprise more than one of a sulforaphane or a derivativethereof. For example, the methods or composition may comprise bothsulforaphane and one or more derivatives thereof, or two or morederivatives. In some embodiments wherein the method or compositioncomprise more than one of a sulforaphane or a derivative thereof, theabove amounts may refer to the amount of each sulforaphane or aderivative thereof, or the total amount of the more than onesulforaphane or derivative thereof.

The term “sulforaphane precursor” refers to any compound, substance ormaterial which can be used to produce sulforaphane. In preferredembodiments, the sulforaphane precursor comprises a compound which canbe converted or metabolized to sulforaphane, preferably by an enzyme. Insome preferred embodiments, the sulforaphane precursor comprisesglucoraphanin. Glucoraphanin is a glucosinolate which is also known as4-methylsulfinylbutyl glucosinolate and1-S-[(1E)-5-(methylsulfinyl)-N-(sulfonatooxy) pentanimidoyl]-1-thio-β-D-glucopyranose.

In some embodiments, the composition comprises about 1 μg to about 10 g,preferably about 250 μg to about 5 g, more preferably about 500 μg toabout 2000 mg, even more preferably about 1 mg to about 750 mg, evenmore preferably about 1.5 mg to about 250 mg, even more preferably about2 mg to about 100 mg, and most preferably about 3 mg to about 75 mg ofthe sulforaphane precursor, preferably glucoraphanin. In someembodiments, compositions suitable for human use comprise about 3.5 mgto about 50 mg of the sulforaphane precursor, preferably glucoraphanin.

In some embodiments, the method comprises administering the sulforaphaneprecursor, preferably glucoraphanin to a subject, in an amount of about1 μg to about 10 g, preferably about 250 μg to about 5 g, morepreferably about 500 μg to about 2000 mg, even more preferably about 1mg to about 750 mg, even more preferably about 1.5 mg to about 250 mg,even more preferably about 2 mg to about 100 mg, and most preferablyabout 3 mg to about 75 mg. In some embodiments wherein the subject is ahuman, the method comprises administration of about 3.5 mg to about 50mg. In some embodiments, the method comprises administering an amount ofsulforaphane precursor to a subject in an amount of about 1 μg/kg toabout 1000 mg/kg, preferably about 5 μg/kg to about 500 mg/kg, morepreferably about 7.5 μg/kg to about 100 mg/kg, even more preferablyabout 10 μg/kg to about 25 mg/kg, and most preferably about 25 μg/kg toabout 10 mg/kg. In some embodiments wherein the subject is a human, themethod comprises administration of about 50 μg/kg to about 800 μg/kg.The above amounts may refer to each dosage administration or a totaldaily dosage.

In some embodiments, the method comprises administration of more thanone sulforaphane precursor. In some embodiments, the compositioncomprises more than sulforaphane precursor. In some embodiments whereinthe method or composition comprises more than one sulforaphaneprecursor, the above amounts may refer to the amount of eachsulforaphane precursor, or the total amount of the sulforaphaneprecursors.

The sulforaphane precursor may be converted or metabolized tosulforaphane. In some embodiments, the sulforaphane precursor isconverted to sulforaphane by an enzyme. In some embodiments, the enzymecapable of converting the sulforaphane precursor to sulforaphanecomprises a glucosidase enzyme, preferably a thioglucosidase enzyme, andmore preferably myrosinase. Myrosinase is also known as thioglucosideglucohydrolase.

In some embodiments, the composition comprises the enzyme in an amountof about 1 pg to about 1 μg, preferably about 50 pg to about 500 ng, andmost preferably about 1 ng to about 150 ng. In some embodiments,compositions suitable for human use comprise about 5 ng to about 75 ngof the enzyme.

In some embodiments, the method comprises administering the enzyme,preferably myrosinase, in an amount of about 1 pg to about 1 μg,preferably about 50 pg to about 500 ng, and most preferably about 1 ngto about 150 ng. In some embodiments wherein the subject is a human, themethod comprises administration of about 5 ng to about 75 ng of theenzyme. In some embodiments, the method comprises administering theenzyme to a subject in an amount of about 0.02 pg/kg to about 0.02ug/kg, preferably about 0.7 pg/kg to about 7 ng/kg, and most preferablyabout 0.02 ng/kg to about 2 ng/kg. In some preferred embodiments whereinthe subject is a human, the method comprises administration of about 0.1ng/kg to about 1 ng/kg. The above amounts may refer to each dosageadministration or a total daily dosage.

In some embodiments, the method comprises administration of more thanone enzyme capable of converting the sulforaphane precursor tosulforaphane. In some embodiments, the composition comprises more thanone enzyme capable of converting the sulforaphane precursor tosulforaphane. In some embodiments wherein the methods or compositionscomprise more than one enzyme, the above amounts may refer to the amountof each enzyme, or the total amount of the enzymes.

The present invention also provides for the use of a broccoli extractand/or powder, including but not limited to broccoli seed and sproutextracts and powders. The present invention provides methods ofadministration of broccoli extract and/or powder, and compositionscomprising broccoli extract and/or powder. In some embodiments, thebroccoli extract or powder is standardized to contain about 1% to about75% w/w, more preferably about 2.5% to about 50%, even more preferablyabout 5% to about 25%, and most preferably about 10% to about 20% of asulforaphane precursor, preferably glucoraphanin. Examples of broccoliextracts and powders include but are not limited to those described inU.S. Pat. Nos. 5,411,986; 5,725,895; 5,968,505; 5,968,567; 6,177,122;6,242,018; 6,521,818; 7,303,770, and 8,124,135, each of which isincorporated by reference in its entirety. Powders of broccoli may beobtained, for example, by air drying, freeze drying, drum drying, spraydrying, heat drying and/or partial vacuum drying broccoli, preferablybroccoli sprouts. In some embodiments, the compositions and methodscomprise use of about 1 μg to about 10 g, more preferably about 250 μgto about 5 g, even more preferably about 500 μg to about 1 g, preferablyabout 600 μg to about 500 mg, more preferably about 750 μg to about 400mg, and most preferably about 1 mg to about 300 mg of the broccoliextract. In some embodiments, the broccoli extract or powder is presentin a composition or administered to a subject in amounts sufficient toprovide a sulforaphane precursor or sulforaphane in the amountsdescribed above. In some embodiments, the composition may furthercomprise an enzyme potentiator, preferably ascorbic acid. In someembodiments, the method may further comprise administration of an enzymepotentiator, preferably ascorbic acid.

The sulforaphane or a derivative thereof, the sulforaphane precursor,and/or the enzyme capable of converting the sulforaphane precursor tosulforaphane may be obtained from any source, including but not limitedto one or more plants from the Brassicaceae (also known as Cruciferae)family Examples of plants from the Brassicaceae family include, but arenot limited to, the following: broccoli, Brussels sprouts, cauliflower,cabbage, horseradish, parsnip, radish, wasabi, watercress, and whitemustard. In some preferred embodiments, sulforaphane precursor,preferably glucoraphanin, and the enzyme, preferably myrosinase, areobtained from broccoli, broccoli sprouts, or broccoli seeds. Thesulforaphane precursor and the enzyme may be obtained from the samesource or from different sources. In some embodiments, both thesulforaphane precursor and the enzyme may be obtained from an extract orpowder from these plants, preferably a broccoli seed or sprout extractor powder.

The present invention provides for the use of an enzyme potentiator orcofactor. Enzyme potentiators may be used to enhance the activity of theenzyme that is capable of converting the sulforaphane precursor tosulforaphane. In some embodiments, the enzyme potentiator comprises anenzyme co-factor, preferably ascorbic acid. Ascorbic acid, also known asascorbate or vitamin C, can potentiate the activity of myrosinase. Insome embodiments, without an enzyme potentiator such as ascorbic acid,the conversion reaction to sulforaphane may be too slow to occur in thelocation needed for peak absorption. The enzyme potentiator may beobtained from a natural source, or it may be produced synthetically.

In some embodiments, the compositions may comprise about 1 mg to about500 mg, preferably about 1 mg to about 250 mg, and most preferably about1 mg to about 125 mg of the enzyme potentiator. In some embodiments,compositions suitable for human use comprise about 1 mg to about 50 mgof the enzyme potentiator.

In some embodiments, the method of the present invention comprisesadministration of an enzyme potentiator, preferably ascorbic acid, in anamount of about 1 mg to about 500 mg, preferably 1 mg to about 250 mg,and most preferably about 1 mg to about 125 mg. In some embodimentswherein the subject is a human, the method comprises administration ofabout 1 mg to about 50 mg. In some embodiments, the method of thepresent invention comprises administration of the enzyme potentiator,preferably ascorbic acid, in an amount of about 0.01 mg/kg to about 3mg/kg, and most about 0.02 mg/kg to about 2 mg/kg. In some embodimentswherein the subject is a human, the method comprises administration ofabout 0.02 mg/kg to about 0.7 mg/kg of the enzyme potentiator. The aboveamounts may refer to each dosage administration or a total daily dosage.

In some embodiments, the method comprises administration of more thanone enzyme potentiator. In some embodiments, the composition comprisesmore than one an enzyme potentiator. In some embodiments wherein themethod or composition comprises more than one enzyme potentiator, theabove amounts may refer to the amount of each enzyme potentiator, or thetotal amount of the enzyme potentiators.

The present invention provides for the use of a mushroom extract orpowder. In some embodiments, the mushrooms may comprise “medicinalmushrooms,” including, but not limited to maitake, shiitake, reishi,cremini, almond, chestnut, wood ear, cloud ear, porcini, ink cap, yartagunbu, enokitake, shemeji, tiger milk, morel, bamboo, golden oyster,pink oyster, king oyster, hiratake, cauliflower, white jelly, goldenjelly, matsutake, Mexican truffle, and straw mushrooms. In preferredembodiments, the mushroom comprises maitake mushroom, shiitake mushroom,reishi mushroom, and/or a mixture of one or more of these.

Maitake mushroom belongs to the species Grifola frondosa. Maitakemushroom may contain a number of fractions having biological activity.Examples of components found in maitake mushroom include but are notlimited to: glucans (such as alpha-glucans and beta-glucans); lipids(such as octadecanoic and octadecadienoic acids); phospholipids (such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol,phosphatidylserine and phosphatidic acid).

Shiitake mushroom belongs to the species Lentinula edodes. Shiitakemushroom may contain a number of fractions having biological activity.Examples of components found in shiitake mushrooms include but are notlimited to: glucans (such as alpha-glucans and beta-glucans), proteins(such as lentin); lipids (such as linoleic acid); and lignins.

Reishi mushrooms belong to the species Ganoderma lucidum. Reishimushrooms may contain a number of fractions having biological activity.Examples of components found in reishi mushroom include but are notlimited to: glucan (such as alpha-glucans and beta-glucans),canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

In some preferred embodiments, the mushroom extract or powder comprisesone or more glucans. A glucan is a polysaccharide of a D-glucose monomerlinked by glycosidic bonds and may be in the alpha or beta form. In someembodiments, the glucan comprises one or more alpha-glucan and/orbeta-glucans. Alpha-glucans include, but are not limited to,1,4-α-glucans and 1,6-α-glucans and beta-glucans include, but are notlimited to, 1,3-β-glucans, 1,4-β-glucans, and 1,6-β-glucans. The glucansmay be expressed in a variety of polymeric configurations. In preferredembodiments, the maitake mushroom extract or powder comprises1,3-β-glucans and/or 1,6-β-glucans. In preferred embodiments, theshiitake mushroom extract or powder comprises 1,4-α-glucans. Inpreferred embodiments, the reishi mushroom extract or powder comprises1,3-β-glucans and/or 1,6-β-glucans. In some embodiments, thecompositions and methods of the present invention may comprise use ofglucans in a purified form or glucans produced synthetically, instead ofa mushroom extract or powder.

In some embodiments, a maitake mushroom extract or powder may be used.In some embodiments, the maitake mushroom extract or powder isstandardized to contain about 1% to about 75%, more preferably about 5%to about 50%, even more preferably about 10% to about 30%, and mostpreferably about 15% to about 20% of one or more glucans, preferablybeta-glucans, and more preferably 1,3-beta glucan and/or1,6-beta-glucan. Examples of maitake mushroom extracts and powdersinclude, but are not limited to, those described in U.S. Pat. No.5,854,404; WO 2007142130, EP 0893449; WO2009063885; WO2006107208;WO2007024496; and WO2001054673, each of which is incorporated byreference in its entirety. Powders of maitake mushroom may be obtained,for example, by air drying, freeze drying, drum drying, spray drying,heat drying and/or partial vacuum drying maitake mushrooms. In someembodiments, the composition comprises about 250 μg to about 100 mg,preferably about 500 μg to about 75 mg, and most preferably about 750 μgto about 50 mg. In some embodiments, compositions suitable for humanscomprise about 1 mg to about 20 mg of maitake mushroom extract. In someembodiments, the method comprises administration of about 250 μg toabout 100 mg, preferably about 500 μg to about 75 mg, and mostpreferably about 750 μg to about 50 mg. In some embodiments wherein thesubject is human, the method comprises administration of about 1 mg toabout 20 mg of maitake mushroom extract. The above amounts may refer toeach dosage administration or a total daily dosage.

In some embodiments, a shiitake mushroom extract or powder may be used.In some embodiments, the shiitake mushroom extract or powder isstandardized to contain about 1% to about 75%, preferably about 10% toabout 60%, even more preferably about 25% to about 50%, and mostpreferably about 30% to about 40% of one or more glucans, preferablyalpha-glucans, and more preferably 1,4-alpha-glucan. Examples ofshiitake mushroom extracts include, but are not limited to, thosedescribed in U.S. Pat. Nos. 5,780,097; 6,582,723; WO2005107496,WO2007024496, and WO2000033069, each of which is incorporated byreference in its entirety. Powders of shiitake mushroom may be obtained,for example, by air drying, freeze drying, drum drying, spray drying,heat drying and/or partial vacuum drying maitake mushrooms. In someembodiments, the composition comprises about 1 mg to about 1 g,preferably about 10 mg to about 500 mg, and most preferably about 25 mgto about 300 mg. In some embodiments, compositions suitable for humanscomprise about 50 mg to about 250 mg of shiitake mushroom extract orpowder. In some embodiments, the method comprises administration ofabout 1 mg to about 1 g, preferably about 10 mg to about 500 mg, andmost preferably about 25 mg to about 300 mg. In some preferredembodiments wherein the subject is human, the method comprisesadministration of about 50 mg to about 250 mg of shiitake mushroomextract or powder to a subject. The above amounts may refer to eachdosage administration or a total daily dosage. The above amounts mayrefer to each dosage administration or a total daily dosage.

In some embodiments, a reishi mushroom extract or powder may be used. Insome embodiments, the reishi mushroom extract comprises about 1% toabout 75%, more preferably about 5% to about 50%, even more preferablyabout 10% to about 30%, and most preferably about 15% to about 20% ofone or more glucans, preferably beta-glucans, and more preferably1,3-beta glucan and/or 1,6-beta-glucan. Powders of reishi mushroom maybe obtained, for example, by air drying, freeze drying, drum drying,spray drying, heat drying and/or partial vacuum drying maitakemushrooms.

In some embodiments, the composition and/or method comprises use of onetype or mushroom extract or powder, such as maitake mushroom extract orpowder, shiitake mushroom extract or powder or reishi mushroom extractor powder. In some embodiments, the composition and/or method comprisesuse of a mixture of one or more types of mushroom extract or powder. Insome embodiments, the composition and/or method comprises use of amixture of one or more of the following: maitake mushroom extract orpowder, shiitake mushroom extract or powder, and reishi mushroom extractor powder. The composition and method may comprise use of an extract ora powder, or a mixture of extracts and powders.

The present invention also provides for the use of any glucan-richcomponent in place of, or in addition to, the mushroom extract orpowder. An example of a glucan-rich component is Baker's yeast. In someembodiments, yeast preparations may be used. In some embodiments, theyeast preparation comprises about 0.1% to about 50%, preferably about0.5% to about 25%, and most preferably about 0.5% to about 10% of one ormore glucans. Examples of yeast preparations include those discussed inU.S. Pat. Nos. 5,223,491 and 5,576,015, each of which is incorporated byreference in its entirety.

The methods of the present invention may further comprise administrationof one or more additional components. The compositions of the presentinvention may further comprise one or more additional components. Theadditional components may include active pharmaceutical ingredients,nutritional supplements, and nutritional extracts. Examples ofadditional components include, but are not limited, ursolic acid,quercetin or a derivative thereof, an aminosugar such as glucosamine, aglycosaminoglycan such as chondroitin, avocado/soybean unsaponifiables,vitamins such as vitamin K2, coffee fruit, magnesium, ursolic acid,proanthocyanidins, alpha- and beta-glucans, curcumin, phytosterols,phytostanols, and S-adenosylmethionine (SAMe). These additionalcomponents may be present in milk thistle (Silybum marianum) extract(silymarin), cranberry (Vaccinium macrocarpon) extract(proanthocyanidins, quercetin, and ursolic acid), turmeric (Curcumalonga).

In some embodiments, the ratio of beta-glucan to sulforaphane or aderivative of (beta-glucan:sulforaphane or a derivative of) is about50:1 to about 1:50, preferably about 25:1 to about 1:25, more preferablyabout 10:1 to about 1:20, more preferably about 5:1 to about 1:10, evenmore preferably about 1:1 to about 1:8, and most preferably about 1:3 toabout 1:5. In some embodiments, the ratio of alpha-glucan tosulforaphane or a derivative of (alpha-glucan:sulforaphane or aderivative of) is about 1:50 to about 50:1, preferably about 1:10 toabout 25:1, more preferably about 1:5 to about 20:1, more preferablyabout 1:1 to about 15:1, even more preferably about 2:1 to about 10:1,and most preferably about 3:1 to about 8:1. In some embodiments, theratio of beta-glucan to sulforaphane precursor of(beta-glucan:sulforaphane precursor) is about 50:1 to about 1:50,preferably about 30:1 to about 1:35, more preferably about 20:1 to about1:25, more preferably about 10:1 to about 1:20, even more preferablyabout 5:1 to about 1:15, and most preferably about 1:1 to about 1:10. Insome embodiments, the ratio of alpha-glucan to precursor(alpha-glucan:precursor) is about 1:50 to about 100:1, preferably about1:25 to about 75:1, more preferably about 1:10 to about 50:1, morepreferably about 1:5 to about 40:1, even more preferably about 1:1 toabout 30:1, and most preferably about 2:1 to about 20:1

In some embodiments, the composition comprises a unit dosage form,including but not limited to pharmaceutical dosage forms suitable fororal, rectal, intravenous, subcutaneous, intramuscular, transdermal,transmucosal, and topical. In some preferred embodiments, thecomposition comprises an orally administrable dosage form or a rectallyadministrable dosage form. Examples of orally administrable dosage formsinclude, but are not limited to a tablet, capsule, powder that can bedispersed in a beverage, a liquid such as a solution, suspension, oremulsion, a soft gel/chew capsule, a chewable bar, or other convenientdosage form known in the art. In preferred embodiments, the compositioncomprises a tablet, capsule, or soft chewable treat. The orallyadministrable dosage forms may be formulated for immediate release,extended release or delayed release.

In some embodiments, at least the sulforaphane precursor, the enzyme,and the enzyme potentiator are provided in a dosage form which allowsfor the release in an area of the gastrointestinal tract having a pH ofat least 4 and preferably at least 5, such as the small intestine,preferably the duodenum. In some embodiments, at least the sulforaphaneor derivative thereof and/or the broccoli extract or powder are providedin a dosage form which allows for the release in an area of thegastrointestinal tract having a pH of at least 4 and preferably at least5, such as the small intestine, preferably the duodenum. In someembodiments, the mushroom extract or powder and/or any optionaladditional components are also released in an area of thegastrointestinal tract having a pH of at least 4 and preferably at least5, such as the small intestine, preferably the duodenum. The smallintestine includes the duodenum, jejunum, and ileum.

In some embodiments, each of these components (i.e, sulforaphaneprecursor, enzyme, enzyme potentiator, sulforaphane or a derivativethereof, broccoli extract or powder, mushroom extract or powder, and/oradditional components) are released simultaneously or concomitantly(i.e., within a short period of time of each other). This providesbenefits over glucoraphanin-containing compositions formulated torelease the glucoraphanin in an area of the gastrointestinal tracthaving a pH below 4, such as the stomach. In low pH environments such asthis, the acidic environment may divert conversion of sulforaphaneprecursor to other, physiologically inactive end products, such assulforaphane nitrile and epithionitrile.

In some embodiments, the compositions may comprise orally administrablecompositions which comprise enteric coated dosage forms or any dosageform which is resistant to degradation in an area of thegastrointestinal tract having pH below 4, such as the stomach. Forexample, the orally administrable composition may comprise a tablet orcapsule comprising an enteric coating. The enteric coating may comprisematerials including, but not limited to cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate,methacrylic acid copolymer, methacrylic acid:acrylic ester copolymer,hydroxypropyl methylcellulose acetate succinate, hydroxypropylmethylcellulose trimellitate, shellac, cellulose acetate trimellitate,carboxymethylethylcellulose, and mixtures thereof. The enteric coatingmay comprise any suitable enteric polymers known in the art. In someembodiments, one or more of the components in the composition may beembedded in a matrix of enteric polymers. In some embodiments, theorally administrable compositions comprise a capsule that dissolvesslowly in gastric acid and travels to the small intestine, such asDRCAPS™ acid resistant capsules, which are marketed by CAPSUGEL® or anyother acid resistant capsules.

In the most preferred form, the orally administrable composition issurrounded by a coating that does not dissolve unless the surroundingmedium is at a pH of at least 4, and more preferably at least 5.Alternatively, a coating may be employed which controls the release bytime, as opposed to pH, with the rate adjusted so that the componentsare not released until after the pH of the gastrointestinal tract hasrisen to at least 4, and more preferably at least 5. Thus, atime-release formulation may be used to prevent gastric presence of thesulforaphane precursor, the enzyme capable of converting thesulforaphane precursor to sulforaphane, and the enzyme potentiator, orof the sulforaphane. The coating layer(s) may be applied onto orallyadministrable composition using standard coating techniques. The entericcoating materials may be dissolved or dispersed in organic or aqueoussolvents. The pH at which the enteric coat will dissolve can becontrolled by a polymer, or combination of polymers, selected and/orratio of pendant groups. For example, dissolution characteristics of thepolymer film can be altered by the ratio of free carboxyl groups toester groups. Enteric coating layers also contain pharmaceuticallyacceptable plasticizers such as triethyl citrate, dibutyl phthalate,triacetin, polyethylene glycols, polysorbates or other plasticizers.Additives such as dispersants, colorants, anti-adhering and anti-foamingagents may also be included.

The compositions may contain one or more non-active pharmaceuticalingredients (also known generally as “excipients”). Non-activeingredients, for example, serve to solubilize, suspend, thicken, dilute,emulsify, stabilize, preserve, protect, color, flavor, and fashion theactive ingredients into an applicable and efficacious preparation thatis safe, convenient, and otherwise acceptable for use. The excipientsare preferably pharmaceutically acceptable excipients. Examples ofclasses of pharmaceutically acceptable excipients include lubricants,buffering agents, stabilizers, blowing agents, pigments, coloringagents, flavoring agents, fillers, bulking agents, fragrances, releasemodifiers, adjuvants, plasticizers, flow accelerators, mold releaseagents, polyols, granulating agents, diluents, binders, buffers,absorbents, glidants, adhesives, anti-adherents, acidulants, softeners,resins, demulcents, solvents, surfactants, emulsifiers, elastomers andmixtures thereof.

In some embodiments, the combination of (i) a sulforaphane precursor,preferably glucoraphanin, (ii) an enzyme capable of converting thesulforaphane precursor to sulforaphane, preferably a glucosidase enzyme,more preferably a thioglucosidase enzyme, and most preferablymyrosinase, (iii) an enzyme potentiator, preferably an enzyme co-factor,more preferably ascorbic acid, and (iv) a mushroom extract or powder(which contains glucans) demonstrates a synergistic effect. In someembodiments, the combination of sulforaphane (or a derivative thereof)and a mushroom extract or powder (which contains glucans) demonstrates asynergistic effect. Synergy refers to the effect wherein a combinationof two or more components provides a result which is greater than thesum of the effects produced by the agents when used alone. In preferredembodiments, the synergistic effect is greater than an additive effect.In some embodiments, the combination of a sulforaphane precursor, anenzyme capable of converting the sulforaphane precursor to sulforaphane,an enzyme potentiator, and a maitake, shiitake, or reishi mushroomextract or powder has a statistically significant, greater effectcompared to: (i) each component alone, (ii) the combination ofsulforaphane precursor and the enzyme alone; and/or (iii) thecombination of sulforaphane precursor, the enzyme, and the enzymepotentiator alone.

In preferred embodiments, the combination of the sulforaphane precursor,the enzyme, the enzyme potentiator, and a mushroom extract or powder(which contains glucans) demonstrates synergy by having a statisticallysignificant and/or greater than additive effect compared to thesulforaphane precursor alone and the mushroom extract or powder alone.In some embodiments, the combination of glucoraphanin, myrosinase,ascorbic acid, and a mushroom extract or powder has a synergistic effectcompared to the combination of glucoraphanin, myrosinase, ascorbic acidalone; and compared to glucans alone.

In some embodiments, the combination of a sulforaphane (or a derivativethereof) and a mushroom extract or powder has a statisticallysignificant and/or greater than additive effect than: (i) sulforaphane(or a derivative thereof) alone, and/or (ii) a mushroom extract orpowder alone. In some embodiments, the combination of sulforaphane andglucan has a synergistic effect compared to sulforaphane alone, andglucan alone.

In some embodiments, the combination of broccoli extract or powder and amushroom extract or powder has a statistically significant and/orgreater than additive effect than: (i) broccoli extract or powder alone,and/or (ii) a mushroom extract or powder alone. In some embodiments, thecombination of broccoli extract or powder and glucan has a synergisticeffect compared to broccoli extract or powder alone, and glucan alone.

The present invention provides methods of use, including methods ofadministration to a subject in need thereof. In some embodiments, themethod comprises administration of the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and a mushroom extract or powder.In some embodiments, the method comprises administration of thecombination of a sulforaphane or a derivative thereof and a mushroomextract or powder. In some embodiments, the method comprisesadministration of the combination of a broccoli extract or powder and amushroom extract or powder.

In some embodiments, the method relates to treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of, cancer, in particular breastcancer, prostate cancer, colon cancer, lung cancer, liver cancer, andbladder cancer in a subject. The methods may be useful in reducingdamage or slowing damage to tissues and organs, such as the breast,prostate, colon, lung, liver, and bladder. The present inventionprovides methods of treating, preventing, decreasing the symptomsassociated with, and/or reducing secondary recurrences of diseases andconditions associated with the reproductive system (including but notlimited to the breast and prostate), colon, liver, bladder, kidney,central nervous system, cardiovascular system, pulmonary system,genitourinary system, hematopoietic system, and joints. The presentinvention also provides for methods of treating, preventing, decreasingthe symptoms associated with, and/or reducing secondary recurrences ofcysts, such as benign cysts.

In some embodiments, the method relates to increasing levels orincreasing gene expression of NAD(P)H:quinone oxidoreductase 1 (NQO-1)in a subject. The method may also be useful in treating, preventing,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of diseases and conditions which would be benefited from anincrease in gene expression or levels of NQO-1. Examples of suchdiseases and conditions include, but are not limited to cancer,myelodysplastic syndrome, cardiovascular disease, and tardivedyskinesia.

In some embodiments, the method relates to treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of a disease or conditionassociated with elevated levels of quinone estrogen. Examples of suchdiseases or conditions include but are not limited to Examples of suchdiseases and conditions include, but are not limited to cancer,myelodysplastic syndrome, cardiovascular disease, and tardivedyskinesia.

In some embodiments, the methods relate to providing a beneficial effecton biomarkers, and treating, preventing, reducing the occurrence of,decreasing the symptoms associated with abnormal levels of thesebiomarkers. Examples of such biomarkers include, but are not limited toNADPH-dependent enzymes, thioredoxin (TXN), thioredoxin reductase-1(Txnrd-1), glutamate-cysteine ligase subunit (GCLC), sulfotransferase1A1 (SULT1A1), heme oxygenase-1 (HMOX1), glutathione peroxidase-3(GPx-3), glutathione S-transferase theta 2 (GSTT2), microsomalglutathione S-transferase 1 (MGST1), aldehyde oxidase (AOX1), aldo-ketoreductase 1B8 (Akr1b8), flavin-containing monooxygenase 2 (FMO2), Fcreceptor region receptor III (Fcgr3), tryptase beta 1 (TPSB1), mast cellprotease-6 (Mcpt6), neurexin-1-alpha (NRXN-1), microphthalmia-associatedtranscription factor (MITF), type II iodothyronine deiodinase (DI02),angiopoietin-14 (Angpt14), cluster of differentiation (CD36), and Nte1.Diseases or conditions associated with elevated or abnormal levels ofthese biomarkers include, but are not limited to cancer, pulmonary andcentral nervous system tuberculosis, multiple sclerosis, Crohn'sdisease, atherosclerosis, osteoarthritis, asthma, stroke, emphysema,diabetic nephropathy, chronic histiocytic intervillositis of theplacenta, hypertension, abdominal aortic aneurysm, inflammatory boweldisease, chronic rhinosinusitis, coronary artery disease, and kidneydisease.

In some embodiments, the method comprises administering to a subject inneed thereof a combination of sulforaphane and a mushroom extract orpowder containing glucan. In some embodiments the method comprisesadministering to a subject in need thereof a combination of broccoliextract or powder and a mushroom extract or powder containing glucan. Insome preferred embodiments, the method comprises administering to thesubject a combination of glucoraphanin, myrosinase, ascorbic acid, and amushroom extract or powder containing glucan. In preferred embodiments,the combinations demonstrate a synergistic effect in the methods of thepresent invention.

In preferred embodiments, one or more components of the combinations(for example, the sulforaphane precursor, the enzyme capable ofconverting the sulforaphane precursor to sulforaphane, the enzymepotentiator, the mushroom extract or powder; or the sulforaphane orderivative thereof and the mushroom extract or powder; or the broccoliextract or powder and the mushroom extract or powder) are administeredtogether in one composition or dosage form, or separately, preferablywithin a period in which their therapeutic properties overlap. In someembodiments, the components of the combinations may be administered intwo or more orally administrable compositions or dosage forms. Forexample, in some embodiments, the sulforaphane precursor, the enzymecapable of converting the sulforaphane precursor to sulforaphane, andthe enzyme potentiator are administered in one orally administrabledosage form, while the a mushroom extract or powder are administered inone or more separate or additional orally administrable dosage form(s).In preferred embodiments, the components of the combination areadministered in one dosage form.

In some embodiments, the combination may be administered at a frequencyof 1 to 10 times daily, preferably 1 to 5 times daily, more preferably 1to 3 times daily, and most preferably 1 time daily.

The dosages disclosed in this application refer preferably to dosagessuitable for humans. Dosage calculations can be determined by those ofskilled in the art by evaluating body weight, surface area, metabolicrate, and species differences.

The term “subject” refers to any animal, including mammals and birds.Mammals include, but are not limited to, humans, dogs, cats, horses,cows, camels, elephants, lions, tigers, bears, seals, and rabbits. Inpreferred embodiments, the subjects comprise mammals that are notconsumed as food, such as humans, cats, and dogs.

EXAMPLES Example 1 (Formulations

The following are exemplary formulation of the present invention:

Formulation A

Glucoraphanin-containing broccoli seed extract (about 12% w/w), 50 mg to5 gramsMyrosinase-containing freeze-dried broccoli sprout powder, 25 mg to 500mgAscorbic acid, 1 mg to 50 mgAlpha Glucan-containing shiitake mushroom extract (about 40% w/w), 1 mgto 250 mg

Formulation B

Glucoraphanin-containing broccoli seed extract (about 12% w/w), 50 mg to5 gramsMyrosinase-containing freeze-dried broccoli sprout powder, 25 mg to 500mgAscorbic acid, 1 mg to 50 mgBeta Glucan-containing maitake mushroom extract (about 20% w/w), 1 to100 mg

Formulation C

An orally administrable composition comprising:Broccoli seed extractBroccoli sprout extractMaitake mushroom extractAscorbic acidHydroxypropylmethyl celluloseMicrocrystalline celluloseCorn starch

Ethylcellulose

Croscarmellose sodiumSodium starch glycolate

Crospovidone

Silicon dioxideSodium alginateMedium chain triglycerides

Maltodextrin Oleic Acid

Magnesium stearateStearic acid

Example 2

A Hydrophobic Interaction Chromatographic (HILIC) method was developed,comprising the following conditions:Column—Waters BEH Amide, 1.7-μm particle size; 2.1 mm×100 mm

Mobile Phase: 20% 10 mM Ammonium Acetate, pH 5.0; 80% Acetonitrile;

Separation mode: isocratic

Column Temperature: 70° C.

Flow Rate: 0.7 mL/min The above conditions allow separation of fivetypical Brassicaceae glucosinolates, including the sulforaphaneprecursor, glucoraphanin.

Example 3 Consumption of Glucoraphanin as a Function of the AscorbicAcid Concentration

About 250 mg of broccoli seed extract containing about 12% (w/w)glucoraphanin were subjected to hydrolysis by a fixed concentration ofbroccoli sprout-derived myrosinase in the presence of variableconcentration of ascorbic acid, ranging from 0 to 600 μmoles/Liter. Thereaction mixtures were thermostated at 38° C.; aliquots were withdrawnevery 15 minutes for 60 minutes, and concentration of glucoraphanindetermined chromatographically. The rate of glucoraphanin consumptionwas interpreted as the rate its conversion to sulforaphane. Graphicalrepresentation of glucoraphanin content reduction as a function ofincreasing ascorbic acid concentration results in a series of linearplots; the slopes of the linear regression lines reflect the rate ofglucoraphanin consumption, in μmoles/minute. It is apparent that in thepresence of 600 μmoles/Liter concentration of ascorbic acid, thereaction rate increased 13-fold relative to that which proceeded in theabsence of modulatory effects of ascorbic acid.

Content of Ascorbic Acid Time, 250 μM min 0 μM 50 μM 125 μM 250 μMFiltered 400 μM 600 μM  0 93.36 93.36 93.36 93.36 93.36 93.36 93.36μmoles 15 92.24 89.20 84.52 80.95 86.31 78.32 75.02 GR 30 90.71 84.2475.92 69.06 79.44 62.78 55.66 45 89.44 80.30 68.09 57.63 71.94 47.6737.50 60 87.79 76.36 59.41 45.76 65.18 33.15 22.09 Slope −0.09293−0.28599 −0.56217 −0.79012 −0.47140 −1.00714 −1.20029 μmol/min Intercept93.496 93.271 93.123 93.053 93.386 93.270 92.734 μmol

Example 4

Equimolar Conversion of Glucoraphanin to Sulforaphane.

A two-part experiment was conducted to further elucidate the role ofascorbic acid in modulating myrosinase activity. All solutions wereprepared in 20 mM Tris-buffered saline, at pH 7.5, previously identifiedas an optimal for myrosinase activity; each sample tube had 100 mg offreeze-dried broccoli powder accurately weighed in as a source ofmyrosinase. Experiment was conducted at 38° C. for 2 hours, with samplealiquots removed in 30-minute increments, and both glucoraphanin andsulforaphane content assessed by HPLC. A strongly acidic “stop” solutionwas utilized to instantaneously inhibit further myrosinase activity inthe removed aliquots. A control sample contained no ascorbic acid, andthe enzymatic conversion proceeded unassisted by a co-factor.

PART 1. In the presence of the fixed concentration of ascorbic acid, 1mmol/Liter, an increasing amount of broccoli seed extract (about 12%glucoraphanin, w/w) was added, ranging from 250 mg to 500 mg.

PART 2. While keeping the amount of broccoli seed extract fixed at 250mg, the concentration of ascorbic acid was varied from 0.4 mmol/Liter to3.8 mmol/Liter.

The table below presents glucoraphanin and sulforaphane expressed inμmoles. It is apparent that within the first 30 minutes in almost allthe reaction mixtures, conversion of glucoraphanin to sulforaphane wascomplete. However, careful examination of the enzymatic conversionoccurring in the control sample, without the stimulating effects ofascorbic acid, reveals an equimolar conversion of glucoraphanin tosulforaphane, i.e., the amount of glucoraphanin consumed results in theequivalent amount of sulforaphane produced.

Glucoraphanin, μmoles Sulforaphane, μmoles Time, min 0 30 60 90 120 0 3060 90 120 GR 250 mg AA 58.02 48.57 37.52 26.58 15.67 3.42 12.08 22.2733.17 42.89 0.0 mM GR 250 mg AA 40.07 21.51 61.95 60.20 60.04 58.25 1.0mM GR 300 mg AA 49.31 24.18 74.40 73.04 72.19 70.56 1.0 mM GR 350 mg AA61.41 25.00 84.92 84.02 83.19 80.02 1.0 mM GR 400 mg AA 71.35 1.56 26.7196.60 95.38 93.39 91.16 1.0 mM GR 500 mg AA 89.41 1.01 33.52 120.16118.45 116.45 112.34 1.0 mM GR 250 mg AA 45.66 15.98 62.06 61.01 60.8858.90 0.4 mM GR 250 mg AA 35.24 26.49 62.19 60.62 60.41 59.10 1.0 mM GR250 mg AA 24.94 36.05 60.85 59.78 59.65 58.08 2.0 mM GR 250 mg AA 22.2438.20 59.95 59.34 58.77 56.99 2.9 mM GR 250 mg AA 21.70 37.87 58.7757.79 58.41 56.17 3.8 mM

In the Part 2 of the experiment, the modulatory effect of the increasingconcentration of ascorbic acid on the activity of myrosinase wasassessed. An initial, apparently linear, increase in myrosinase-promotedconversion of glucoraphanin to sulforaphane is observed to about 2mmol/L of ascorbic acid concentration, followed subsequently by aconsiderable leveling off.

Finally, examination of sulforaphane yield of after 30 minutes withinthe PART 1 of the experiment, reveals that in the presence of 1mmol/Liter of ascorbic acid, the fixed amount of myrosinase contained in100 mg of freeze-dried broccoli sprout powder is capable of generatingat least 200 μmoles of sulforaphane, in a predictably linear fashion.FIGS. 1, 2, 3, and 4 demonstrate the results of this study.

Example 5

Conversion of Glucoraphanin to Sulforaphane in the Presence of SimulatedIntestinal Fluid.

Simulated Intestinal Fluid (SIF) powder, a commercially suppliedconcentrate closely approximating the human intestinal content in termsof composition, pH and ionic strength, was used. The experiment utilizeda USP Dissolution Apparatus 2 (paddles), where into six dissolutionvessels 500 mL of Simulated Intestinal Fluid was dispensed, along with150 mg of freeze-dried broccoli sprout powder as a source of myrosinase.In vessels 1-4, the concentration of ascorbic acid was varied from 0.25to 1.00 mmol/Liter; in vessel 5, in addition to 1 mmol/Liter ascorbicacid, 3.125 g of pancreatin (8×USP) was suspended; in vessel 6, inaddition to 1 mmol/Liter ascorbic acid, and 3.125 g of pancreatin(8×USP), a doubled amount of freeze-dried broccoli sprout powder (300mg) was added. After vessels were brought to 38° C., 250 mg ofglucoraphanin-rich (12%, w/w) broccoli seed extract was added to each,and the resulting suspensions were stirred at 75 RPM for 2 hours.Aliquots were withdrawn every 15 minutes and assayed for sulforaphane.FIG. 4 shows direct correlation between larger yield of sulforaphane andhigher concentrations of ascorbic acid, especially at the earlier stagesof the experiment.

Example 6

The following study was conducted to determine the effect of thecombination of sulforaphane and a maitake mushroom extract containing20% β-glucans on gene expression of Nad(P)H:quinone oxidoreductase 1(NQO-1). NQO-1 encodes a protein that is able to metabolize estrogenquinones, preventing them from forming DNA adducts that cause mutationsand ultimately carcinogenesis. An increase in NQO-1 expression isfavorable for breast, colon, liver, lung, skin and prostate health.

In the study, the macrophage cell line RAW 264.7 was treated with DMSO(vehicle control), sulforaphane (SFN), maitake mushroom extract havingabout 20% beta-glucan content (Maitake), or the combination ofsulforaphane and maitake mushroom extract, for 24 hours. In particular,the cells were treated with one of the following: (i) DMSO (vehiclecontrol), (ii) 0.5 μM SFN, (iii) 250 μg/mL Maitake, (iv) 500 μg/mLMaitake, (v) 750 μg/mL Maitake, (vi) 0.5 μM SFN and 250 μg/mL Maitake,(vii) 0.5 μM SFN and 500 μg/mL Maitake, and (viii) 0.5 μM SFN and 750μg/mL Maitake. Gene expression of NQO-1 gene expression was analyzed viaquantitative RT-PCR. The results, which are depicted in FIG. 5, show thefollowing:

Fold Increase in Treatment NQO-1 gene expression DMSO 1.00 0.5 μM SFN12.96 250 μg/mL Maitake 1.34 0.5 μM SFN + 250 μg/mL 55.97 Maitake 500μg/mL Maitake 1.67 0.5 μM SFN + 500 μg/mL 60.52 Maitake 750 μg/mLMaitake 2.31 0.5 μM SFN + 750 μg/mL 59.47 Maitake

The results demonstrate that the combination of sulforaphane and maitakemushroom extract had a synergistic effect compared to each componentalone. This effect was found to be more than merely additive.

Example 7

The following study was conducted to determine the effect of thecombination of sulforaphane and a shiitake mushroom extract containing40% alpha glucans on gene expression of Nad(P)H:quinonequinoneoxidoreductase 1 (NQO-1).

In the study, the macrophage cell line RAW 264.7 was treated with DMSO(vehicle control), sulforaphane (SFN), shiitake mushroom extract havingat least 20% alpha-glucan content (Shiitake), or the combination ofsulforaphane and shiitake mushroom extract, for 24 hours. In particular,the cells were treated with one of the following: (i) DMSO (vehiclecontrol), (ii) 0.5 μM SFN, (iii) 100 μg/mL Shiitake, (iv) 250 μg/mLShiitake, (v) 500 μg/mL Shiitake, (vi) 0.5 μM SFN and 100 μg/mLShiitake, (vii) 0.5 μM SFN and 250 μg/mL Shiitake, and (viii) 0.5 μM SFNand 500 μg/mL Shiitake. Gene expression of NQO-1 gene expression wasanalyzed via quantitative RT-PCR. The results, which are depicted inFIG. 6, show the following:

Fold Increase in Treatment NQO-1 gene expression DMSO 1.00 0.5 μM SFN12.96 100 μg/mL Shiitake 1.00 0.5 μM SFN + 100 μg/mL 14.58 Shiitake 250μg/mL Shiitake 1.27 0.5 μM SFN + 250 μg/mL 16.07 Shiitake 500 μg/mLShiitake 1.26 0.5 μM SFN + 500 μg/mL 15.20 Shiitake

The results demonstrate that the combination of sulforaphane andshiitake mushroom extract had a synergistic effect compared to eachcomponent alone. This effect was found to be more than merely additive.

Example 8

A subject presents with breast cancer and is suffering from symptomsincluding damaged breast tissue and breast pain. She is administered atablet containing glucoraphanin, myrosinase, ascorbic acid, and amaitake mushroom extract. The tablet is an enteric coated formulationwhich releases the contents in the small intestine. After one month ofdaily administration of the tablet, the subject experiences modulationof surrogate biomarkers including NQO-1 which correlate with improved insymptoms.

Example 9

A subject presents with breast cancer and is suffering from symptomsincluding damaged breast tissue and breast pain. She is administered atablet containing glucoraphanin, myrosinase, ascorbic acid, and ashiitake mushroom extract. The tablet is an enteric coated formulationwhich releases the contents in the small intestine. After one month ofdaily administration of the tablet, the subject experiences modulationof surrogate biomarkers including NQO-1 which correlate with improvementin symptoms.

What is claimed:
 1. An orally administrable composition comprising acombination of: a sulforaphane precursor; a glucosidase enzyme capableof converting the sulforaphane precursor to sulforaphane; a glucosidaseenzyme cofactor; and a glucan; the combination providing asynergistically effective ratio of sulforaphane precursor:glucan of fromabout 100:1 to about 1:50.
 2. The orally administrable composition ofclaim 1, wherein the sulforaphane precursor comprises glucoraphanin, theglucosidase enzyme comprises myrosinase, and the glucosidase enzymecofactor comprises ascorbic acid.
 3. The orally administrablecomposition of claim 1, wherein the composition comprises anenteric-coated dosage form.
 4. The orally administrable composition ofclaim 1, wherein the composition comprises a mushroom extract or powdercomprising the glucan.
 5. The orally administrable composition of claim1, wherein the composition comprises a yeast extract or powdercomprising the glucan.
 6. The orally administrable composition of claim1, wherein the glucan is a β-glucan.
 7. The orally administrablecomposition of claim 1, wherein the glucan is an α-glucan.
 8. The orallyadministrable composition of claim 4, wherein the mushroom extract orpowder is derived from one or more of a maitake mushroom, a shiitakemushroom, and a reishi mushroom.
 9. The orally administrable compositionof claim 5, wherein the yeast extract or powder is derived from abaker's yeast.
 10. The orally administrable composition of claim 1,wherein the composition further comprises one or more additionalcomponents selected from the group consisting of quercetin, anaminosugar, a glycosaminoglycan, avocado/soybean unsaponifiables, avitamin, a coffee fruit, magnesium, silymarin, proanthocyanidins,ursolic acid, curcumin, phytosterols, and phytostanols.
 11. The orallyadministrable composition of claim 1, wherein the composition comprisesglucoraphanin, myrosinase, ascorbic acid, and β-glucan.
 12. The orallyadministrable composition of claim 1, wherein the composition comprisesglucoraphanin, myrosinase, ascorbic acid, and α-glucan.
 13. The orallyadministrable composition of claim 8, wherein the composition comprisesglucoraphanin, myrosinase, ascorbic acid, and maitake mushroom extractor powder.
 14. The orally administrable composition of claim 8, whereinthe composition comprises glucoraphanin, myrosinase, ascorbic acid,maitake mushroom extract or powder, and shiitake mushroom extract orpowder.
 15. The orally administrable composition of claim 1, wherein thecomposition comprises a broccoli extract or powder comprising thesulforaphane precursor.
 16. An orally administrable compositioncomprising a combination of: a sulforaphane or sulforaphane derivative;and a glucan; the combination providing a synergistically effectiveratio of sulforaphane or sulforaphane derivative:glucan of from about100:1 to about 1:50.
 17. The orally administrable composition of claim16, wherein the composition comprises an enteric-coated dosage form. 18.The orally administrable composition of claim 16, wherein thecomposition comprises a mushroom extract or powder comprising theglucan.
 19. The orally administrable composition of claim 16, whereinthe composition comprises a yeast extract or powder comprising theglucan.
 20. The orally administrable composition of claim 16, whereinthe glucan is a β-glucan.
 21. The orally administrable composition ofclaim 16, wherein the glucan is an α-glucan.
 22. The orallyadministrable composition of claim 18, wherein the mushroom extract orpowder is derived from one or more of a maitake mushroom, a shiitakemushroom, and a reishi mushroom.
 23. The orally administrablecomposition of claim 19, wherein the yeast extract or powder is derivedfrom a baker's yeast.
 24. The orally administrable composition of claim16, wherein the composition further comprises one or more additionalcomponents selected from the group consisting of quercetin, anaminosugar, a glycosaminoglycan, avocado/soybean unsaponifiables, avitamin, a coffee fruit, magnesium, silymarin, proanthocyanidins,ursolic acid, curcumin, phytosterols, and phytostanols.
 25. The orallyadministrable composition of claim 16, comprising sulforaphane orsulforaphane derivative and β-glucan.
 26. The orally administrablecomposition of claim 16, comprising sulforaphane or sulforaphanederivative and α-glucan.
 27. The orally administrable composition ofclaim 22, wherein the composition comprises sulforaphane or sulforaphanederivative and maitake mushroom extract or powder.
 28. The orallyadministrable composition of claim 22, wherein the composition comprisessulforaphane or sulforaphane derivative, maitake mushroom extract orpowder, and shiitake mushroom extract or powder.
 29. The orallyadministrable composition of claim 16, wherein the composition comprisesa broccoli extract or powder comprising the sulforaphane or sulforaphanederivative.